In the past few years, studies of the spatial and temporal aspects of T cell signaling have revolutionized our view of how T cells respond to foreign antigens. These studies have primarily focused on mature T cells signaling, while our understanding of the spatial and temporal aspects of thymocyte signaling have lagged behind. We propose to address this area by employing2-photon laser scanning microscopy, in conjunction with fetal thymic organ culture, to analyze the cellular and molecular events that occur during positive and negative selection. Aim 1 describes experiments to analyze the cellular events associated with various forms of thymic selection. We have previously observed distinct types of interactions between thymocytes and positive selecting thymic stromal cells, including stable and dynamic contacts, partial engulfment of thymocytes by stromal cells, and indications of thymocyte apoptosis. We will now ask whether these behaviors coincide with different forms of selection such as: negative selection, early or late positive selection, and positive selection via MHC class I or II. Aim 2 describes experiments to analyze molecular rearrangements during thymic selection. We will use both Immunofluorescence analysis of fixed samples and real-time analysis in thymic organ cultures. We will use fluorescent fusion proteins as markers for TCR signaling components and calcium sensitive dyes to analyze changes in intracellular calcium concentration during thymic selection. These studies should allow us to address the role of spatial and temporal patterns of TCR signaling in determining the outcome of thymic development. This fundamental information about the process whereby immature thymic precursors give rise to mature, functional T cells, will improve our ability to manipulate he immune response to disease and to improve human health.